Department of Laboratory Animal Science, Kitasato University School of Medicine, Sagamihara, 252-0374, Japan.
Center for Genetic Studies of Integrated Biological Functions, Kitasato University School of Medicine, Sagamihara, 252-0374, Japan.
Sci Rep. 2022 Sep 2;12(1):14923. doi: 10.1038/s41598-022-19153-0.
TAR DNA-binding protein 43 kDa (TDP-43), a nuclear protein, plays an important role in the molecular pathogenesis of amyotrophic lateral sclerosis (ALS). The long-disordered C-terminal region (CTR) of TDP-43 is known to be aggregation-prone and a hotspot for ALS mutations, so elucidation of the physiological function of CTR will provide insights into the pathogenesis of ALS. The CTR has two Gly, aromatic, and Ser-rich (GaroS) segments and an amyloidogenic core divided into a hydrophobic patch (HP) and a Gln/Asn (Q/N)-rich segment. Although TDP-43 lacking the CTR is known to be unstable, as observed in knock-in mice, it is unclear which of these segments contributes to the stability of TDP-43. Here, we generated 12 mouse lines lacking the various sub-regions of CTR by genome editing and compared the embryonic lethality of homozygotes, and protein and mRNA expression levels of TDP-43. We demonstrated the functional diversity of the four segments of CTR, finding that the presence of the Q/N-rich segment greatly restored the protein stability of TDP-43. In addition, we found that the second GaroS deletion did not affect protein stability and mouse development.
TAR DNA 结合蛋白 43kDa（TDP-43）是一种核蛋白，在肌萎缩侧索硬化症（ALS）的分子发病机制中发挥重要作用。TDP-43 的长无序 C 端区域（CTR）已知易于聚集，是 ALS 突变的热点，因此阐明 CTR 的生理功能将为 ALS 的发病机制提供深入了解。CTR 有两个 Gly、芳香族和 Ser 丰富（GaroS）片段和一个由疏水区（HP）和 Gln/Asn（Q/N）丰富片段组成的淀粉样核心。尽管缺乏 CTR 的 TDP-43 如 knock-in 小鼠中观察到的那样不稳定，但尚不清楚这些片段中的哪一个有助于 TDP-43 的稳定性。在这里，我们通过基因组编辑生成了 12 条缺乏 CTR 各种亚区域的小鼠系，并比较了纯合子的胚胎致死率以及 TDP-43 的蛋白和 mRNA 表达水平。我们证明了 CTR 的四个片段的功能多样性，发现富含 Q/N 的片段的存在极大地恢复了 TDP-43 的蛋白稳定性。此外，我们发现第二个 GaroS 缺失不影响蛋白稳定性和小鼠发育。
